Lilium regale wilson LrPAL-1 gene and application thereof

A gene and lily technology is applied to the Minjiang lily LrPAL-1 gene and its application field to achieve the effects of improving resistance to pathogenic fungi, increasing the degree of lignification, strong application prospects and promotion value

Active Publication Date: 2020-03-31
YANGTZE NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, there is no report about the simultaneous participation of lil...

Method used

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  • Lilium regale wilson LrPAL-1 gene and application thereof
  • Lilium regale wilson LrPAL-1 gene and application thereof
  • Lilium regale wilson LrPAL-1 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Minjiang Lily LrPAL-1 Full-length gene cloning and sequence analysis

[0035] Botrytis cinerea ( Botrytis cinerea ) spore solution to infect the leaves of Minjiang Lilium (sprouting stage). The leaves at different times (6h, 12, 24h and 48h) were taken as the experimental group, and the leaves treated with no spore solution were taken as the control group. Using TRIzol™Plus RNA Purification Kit (12183555, Invitrogen™), extract the total RNA according to the instructions, use DNase I (18047019, Invitrogen™) to remove the residual trace DNA, and use a differential spectrophotometer to measure the concentration of RNA, and a part of high-quality Some of the RNA was sent to BGI’s BIOSEQ500 sequencing platform for sequencing analysis; the other part of the RNA was kept for future use.

[0036] Through transcriptome sequencing analysis, three genes encoding phenylalanine ammonia-lyase from Lily of Minjiang River ( LrPAL ) Unigene (Unigene3248_All, Unigene14345_...

Embodiment 2

[0046] Example 2 Construction of Plant Overexpression Vectors

[0047] (1) Construction of pART-CAM::LrPAL-1 overexpression vector

[0048] Minjiang lily LrPAL-1 For the full-length gene sequence (SEQ ID NO.1), design primers LrPAL-1-inf-F and LrPAL-1-inf-R, and introduce seamless cloning (In-fusion) vector linker sequences into the primers. Using the positive clone plasmid connected by TA in Example 1 as a template, and using LrPAL-1-inf-F and LrPAL-1-inf-R as primers to carry out gene LrPAL-1 specific amplification.

[0049] The primer sequences are as follows:

[0050] LrPAL-1-inf-F: 5’- GGAGAGGACACGCTCGAG ATGGCATCCAAGGACAGCT-3'

[0051] LrPAL-1-inf-R: 5’- TTAAAGCAGGACTCTAGA TCAGAGATTCCAGTACCCCCT-3'

[0052] Among them, the underline is the linker sequence of the In-fusion cloning vector.

[0053] PCR amplification system: 2xfast pfu master Max 10ul, forward primer (LrPAL-1-F, 10μM) 1μL, reverse primer (LrPAL-1-R, 10μM) 1μL, template (DNA) 1μL, sterile ddH 2 O to...

Embodiment 3

[0061] Example 3 Agrobacterium-mediated transformation of tobacco and screening of transgenic positive lines

[0062] Tobacco ( Nicotiana tabacum cv. Petit Havana SR1) seeds were rinsed with sterile water, sterilized with 75% ethanol for 1 min, sterilized with sodium hypochlorite solution (2% available chlorine) for 15 min, rinsed with sterile water for 3-5 times, and dried with sterile filter paper. Water, inoculated on LS medium (pH 5.8).

[0063] Streak the glycerol bacteria on YEB solid medium (50 μg / mL streptomycin + 20 μg / mL rifampicin), and after culturing at 28 °C for two days, pick a single clone and inoculate it into 5 ml of YEB liquid medium (50 μg / mL streptomycin). Mycin+20μg / mL rifampicin), 28℃, 220rpm constant temperature shaking culture overnight to OD 600 At about 0.6, centrifuge at 5000rpm for 10min to collect the bacteria, and resuspend the collected bacteria to OD with MS liquid medium 600 =0.4.

[0064] After 1 to 2 months, take the aseptic tender lea...

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Abstract

The invention discloses a lilium regale wilson LrPAL-1 gene and application thereof. The nucleotide sequence of the LrPAL-1 gene is as shown in SEQ ID NO.1 and the coded amino acid sequence of the LrPAL-1 gene is as shown in SEQ ID NO.2. The overexpression vector containing the LrPAL-1 gene is transferred into tobacco, the lignification degree of an obtained transgenic tobacco plant is increased,the lodging resistance of the plant is improved, and particularly, the upright state of fresh cut lily flowers can be maintained. The transgenic plant has an obvious inhibition effect on pathogenic bacteria botrytis cinerea and alternaria alternata. Meanwhile, the functional genomics researches prove that the lilium regale wilson LrPAL-1 gene has an important biological function of changing the morphology of plant leaves. The invention provides an important target for molecular genetic improvement of lilium plants; the lilium regale wilson LrPAL-1 gene has an important value and significance for cultivation and production of new varieties of plants with ornamental value and landscape ecological value; and a technical support is provided for cultivation of new lilium germplasm with dual values of lodging resistance and pathogenic bacteria resistance.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a Minjiang lily LrPAL-1 Genes and their applications. Background technique [0002] Lily is a perennial herbaceous bulbous plant that is distributed all over the world. As one of the important ornamental flowers in the world, lily has important economic value. my country is the distribution center of lily in the world, and has abundant wild lily resources, with about 55 species (Flora of China 24: 135–149. 2000.). Minjiang lily ( Lilium regale Wilson) is an important wild germplasm resource in Liliaceae Lilium, which is widely distributed in the Minjiang River Basin in Sichuan, my country (Yang Liping and Fu Yongyao, Research on the Utilization of Lily Resources in China [M], Harbin: Northeast Forestry University Press, 2018.11). Compared with model plants, the growth and development regulation mechanism of Lily Minjiang is complex and specific. Previous s...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/82C12N15/84A01H5/00A01H5/12A01H6/82
CPCC12N9/88C12N15/8282C12N15/8261C12Y403/01005Y02A40/146
Inventor 符勇耀杨利平张雪梅杨韦徐文姬
Owner YANGTZE NORMAL UNIVERSITY
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